Project Summary Herpes simplex virus 1 (HSV-1) infections cause a variety of diseases in humans, ranging from cold sores to blinding ocular infections and life-threatening encephalitis. The virus has two distinct phases of its life cycle: lytic and latent infections. Lytic infection occurs in fibroblasts and epithelial cells and some types of neurons. Latent infection, which only occurs in neurons, represses overall gene expression with no infectious virus produced. Infected cell protein 0 (ICP0), expressed by HSV-1 during lytic infection, plays a pivotal role in the switch between lytic and latent infections. ICP0 is required for efficient viral replication, strongly stimulating viral transcription via its E3 ubiquitin (Ub) ligase activity. E3 Ub ligases facilitate the attachment and polymerization of the post-translational modification, Ub, to target proteins, marking them for degradation by the proteasome. Recent studies suggest that ICP0 directs ubiquitination of selected host cell proteins that restrict viral gene expression in epithelial cells and fibroblasts, thus leading to robust lytic infection. In neurons, however, where neuronal proteins restrict viral gene expression to drive the establishment of latency, the targets of ICP0-mediated ubiquitination and degradation are unknown. Efforts to identify ICP0-specific targets have been challenging given the limitations in the purity and detection of proteins via traditional methods. The unknown identity of the full repertoire of cellular proteins targeted for ICP0 ubiquitination and degradation represents a critical barrier to understanding its function, since these target proteins have the potential to restrict lytic infection. The long-term goal of our research is to understand how virus-host interactions regulate the HSV replication cycle. The objective of this application is to use an advanced proteomics approach to answer the question: Which unique neuronal factors does ICP0 ubiquitinate and degrade to lift the restriction on viral gene expression and replication? To address this question, we will use a method to enrich for ubiquitinated proteins, followed by tandem mass spectrometry (TMS) to identify novel targets of ICP0 ubiquitination. The rationale for carrying out these experiments is that by understanding how ICP0 contributes to lytic infection in neurons, it may be possible to develop anti-HSV therapies. To accomplish this objective, the proposal brings together two investigators with distinct and synergistic expertise: Dr. Bertke (neurovirology) and Dr. Davido (HSV-1 ICP0 function). With this background the following Specific Aims are proposed: Aim #1: Identify new cellular targets that are ubiquitinated by HSV-1 ICP0 in primary adult neurons. Aim #2: Determine the role of novel and candidate cellular targets of ICP0-mediated ubiquitination in neuronal infection. This proposal will contribute to identifying new cellular proteins involved in the biology of ICP0 using an innovative approach, which can be applied to isolate proteins and pathways that play important roles in viral infections and protein stability in neurons.